Myocardial β(2) -adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure

BACKGROUND AND PURPOSE

We investigated whether β₂-adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart.

EXPERIMENTAL APPROACH

We explored the angiogenic effects of β₂-adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β₂-adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β₂-adrenoceptor −/− mice undergoing MI.

KEY RESULTS

Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β₂-adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-β₂-adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, β₂-adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β₂-adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In β₂-adrenoceptor−/− mice, we found a ∼25% reduction in cardiac capillary density compared with β₂-adrenoceptor+/+ mice. The lack of β₂-adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls.

CONCLUSIONS AND IMPLICATION

β₂-Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism.     

Effect of captopril on myocardial β-adrenoceptor density and Giα-proteins in patients with mild to moderate heart failure due to dilated cardiomyopathy

In end-stage heart failure due to idiopathic dilated cardiomyopathy ₁-adrenoceptors are downregulated and G₁-proteins are upregulated. The aim of the present study was to investigate the influence of the angiotensin-converting enzyme inhibitor captopril on -adrenoceptor density and G_i-proteins in sequential endomyocardial biopsies. Nineteen patients with mild to moderate congestive heart failure due to idiopathic dilated cardiomyopathy (NYHA Class II–III) were studied before and after 8–11 weeks of therapy. Patients were randomised into a captopril and a control group; 9 patients received captopril 12.5–50 mg per day, (divided in 2–3 doses) p.o. in addition to conventional therapy with digoxin and diuretics, and 10 controls received conventional therapy only. Echocardiography, spiroergometry, right heart catheterisation and endomyocardial biopsies were performed before (baseline) and after treatment. Compared to baseline, captopril increased total -adrenoceptor density by selectively increasing ₁-adrenoceptors (31.6 vs 41.2 fmol·mg^–1; p<0.05) but had no significant effect on G_i-proteins. The results indicate that treatment with angiotensin-converting enzyme inhibitors partly restores myocardial ₁-adrenoceptor density, and this action effect may contribute to the clinical improvement of patients with idiopathic dilated cardiomyopathy treated in this way.     

Evolving concepts concerning cardiac β-adrenoceptor function in heart failure

This review describes the clinical application of β-adrenergic blocking drugs over time in patients with congestive heart failure. The different pharmacological properties of these drugs in relation to clinical outcomes raises questions as to their optimal desirable profile based on studies showing the undesirable effects of excessive β(2)-AR activation. The rationale for the use of selective β(2)-AR antagonists in heart failure is discussed.     

Chronic heart failure: β-blockers and pharmacogenetics

Purpose  The European Society of Cardiology recommends that β-blockers should be considered for treating all patients with stable, mild, moderate, or severe heart failure (HF) who are receiving standard treatment, unless there is a contraindication. Despite the significant benefit of the drug, there is widespread recognition of patient-to-patient variability in drug response. The genetic determinants of responses to drugs have important implications for the clinical course and management of HF. Pharmacogenetics (PGt) has drawn great attention for its potential to redirect personal care and public health paradigms. The aim of this review was to gather information on PGt of β-blockers in HF treatment. Methods  We searched for articles related to PGt of β-blockers in the PubMed database and attempted to cover all related articles. Results  Several genetic polymorphisms affecting proteins in the β-adrenergic receptor signaling pathway have been proposed as modifiers of HF risk. The most relevant of these to this review is the pharmacogenetic interactions between the genetic variants of catecholamine receptors or their effectors and β-blockade for the treatment of HF. Conclusions  Interindividual variability of responsiveness to β-blockers can be explained by PGt data of adrenaline-related genes. To demonstrate that pharmacogenetic intervention produces successful individualized β-blocker treatment for HF patients, prospective, randomized, and pharmacogenomics (PGx)-based clinical trials are required. Our assessment is that we are already at a turning point in the history of clinical pharmacology.     

β-adrenoceptor blocker treatment and the cardiac β-adrenoceptor-G-protein(s)-adenylyl cyclase system in chronic heart failure

Recent studies showed that chronic β-adrenoceptor (AR) blocker treatment exerts beneficial effects in patients with chronic heart failure (CHF). In CHF, sympathetic drive to the heart is increased, and this causes pathological changes in cardiac β-AR-G-protein(s)-adenylyl cyclase system: Cardiac β-1 AR are decreased, and amount and activity of cardiac G_i-protein and G-protein-coupled receptor kinase (GRK) are increased resulting in diminished cardiac β-AR functional responsiveness. One possible mechanism of beneficial effects of β-AR blockers could be that they prevent adverse effects of increased sympathetic activity and up-regulate cardiac (and vascular) β-AR density, and by this, enhance β-AR-mediated effects. Another possibility could be that chronic β-AR blocker treatment normalizes activity of G_i-protein and may thereby restore β-AR functional responsiveness. Moreover, failing human heart exhibits an inverse force–frequency relationship. β-AR blockers reduce heart rate; this may, therefore, improve force of contraction. One of the strongest stimuli to activate GRK is increased sympathetic activity (as in CHF) via β-AR stimulation. β-AR blockers, by blocking β-AR, can prevent GRK activation and/or can reduce the (previously enhanced) GRK activity, and this might—at least partly—contribute to beneficial effects of β-AR blockers in CHF treatment. Finally, the “loss-of-function” Arg389Gly β-1 AR polymorphism seems to determine heart rate and blood pressure responses to β-1 AR blocker administration: Arg389Arg β-1 AR subjects exhibit stronger effects than subjects with one or two Gly389 alleles. Thus, it might be predicted that patients homozygous Arg389 β-1 AR should be good responders, whereas patients homozygous Gly389 β-1 AR polymorphism should be poor or non-responders. Dedicated to Professor Dr. Karl-Heinz Jakobs at the occasion of his 65th birthday.     

The design, synthesis and pharmacological characterization of novel β₂-adrenoceptor antagonists

BACKGROUND AND PURPOSE

Selective and potent antagonists for the β₂-adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology.

EXPERIMENTAL APPROACH

A range of pharmacological assays was used to assess potency, affinity, selectivity (β₂-adrenoceptor vs. β₁-adrenoceptor) and efficacy.

KEY RESULTS

Ten novel compounds were identified but none had as high affinity as the prototypical β₂-adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β₂-adrenoceptors. Most of the ligands were inverse agonists for β₂-adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β₂-adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β₂-adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour.

CONCLUSIONS AND IMPLICATIONS

In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology.     

AMPK-mediated GSK3β inhibition by isoliquiritigenin contributes to protecting mitochondria against iron-catalyzed oxidative stress

Isoliquiritigenin (ILQ), a flavonoid compound originated from Glycyrrhiza species, is known to activate SIRT1. Arachidonic acid (AA) in combination with iron (a catalyst of auto-oxidation) leads cells to produce excess reactive species with a change in mitochondrial permeability transition. In view of the importance of oxidative stress in cell death and inflammation, this study investigated the potential of ILQ to protect cells against the mitochondrial impairment induced by AA + iron and the underlying basis for this cytoprotection. Treatment with ILQ inhibited apoptosis induced by AA + iron, as evidenced by alterations in the levels of the proteins associated with cell viability: ILQ prevented a decrease in Bcl-x_L, and cleavage of poly(ADP-ribose)polymerase and procaspase-3. Moreover, ILQ inhibited the ability of AA + iron to elicit mitochondrial dysfunction. In addition, superoxide generation in mitochondria was attenuated by ILQ treatment. Consistently, ILQ prevented cellular H₂O₂ production increased by AA + iron, thereby enabling cells to restore GSH content. ILQ treatment enhanced inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β), and prevented a decrease in the GSK3β phosphorylation elicited by AA + iron, which contributed to protecting cells and mitochondria. GSK3β phosphorylation by ILQ was preceded by AMP-activated protein kinase (AMPK) activation, which was also responsible for mitochondrial protection, as shown by reversal of its effect in the experiments using a dominant negative mutant of AMPK and compound C. Moreover, the AMPK activation led to GSK3β phosphorylation. These results demonstrate that ILQ has the ability to protect cells from AA + iron-induced H₂O₂ production and mitochondrial dysfunction, which is mediated with GSK3β phosphorylation downstream of AMPK.     

Chronic β-adrenoceptor antagonist treatment controls cardiovascular remodeling in heart failure in the aging spontaneously hypertensive rat

Selective β1-adrenoceptor antagonists are part of standard therapy to prolong survival in human heart failure. This study has measured structural, functional, and electrical changes in the cardiovascular system of aging male spontaneously hypertensive rats (SHRs) to determine whether β1-adrenoceptor antagonist treatment can prevent or reverse the development of cardiovascular remodeling and heart failure in these rats. Fifteen-month-old male Wistar-Kyoto (WKY) rats or SHRs were treated with increasing metoprolol doses (30 mg·kg·d for 4 weeks, then 50 mg·kg·d for 4 weeks, then 80 mg·kg·d for 16 weeks po). Cardiovascular structure and function were determined using organ wet weight, in vivo echocardiography, histological analysis of inflammation and collagen, isolated heart and thoracic aortic ring preparations, and single cell microelectrode measurements. From 15 months, untreated SHRs developed left ventricular dilation, hypertrophy, inflammatory cell infiltration and fibrosis, and action potential prolongation together with progressive systolic, diastolic, and endothelial dysfunction and increased cardiac stiffness. Treatment with metoprolol decreased systolic blood pressure at 21 months only but improved survival, decreased ventricular weight, prevented chamber dilation, reduced inflammation, decreased fibrosis, attenuated action potential prolongation, improved systolic and diastolic function, decreased stiffness and improved endothelium-independent vascular responses. Chronic metoprolol treatment markedly attenuated both cardiac and vascular remodeling in the aging SHRs, thus attenuating the onset of heart failure and improving survival, independent of blood pressure reduction.     

Effect of β-N-methylamino-l-alanine on oxidative stress of liver and kidney in rat

β-N-methylamino-l-alanine (l-BMAA) is a neurotoxic amino acid, found in the majority of cyanbacterial genera tested. Evidence for implication of l-BMAA in neurodegenerative disorders, like amyotrophic lateral sclerosis (ALS), relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. The involvement of l-BMAA in oxidative stress was demonstrated in several studies in the central nervous system. In the present study, we investigated the effect of l-BMAA on the oxidative stress responses of liver and kidney in rats treated by intraperitoneal administration with this amino acid. Oxidative stress was demonstrated by the quantification of lipid peroxidation, the measurement of both catalase and glutathione peroxidase activities, as well as the quantification of glutathione (GSH) levels and the total antioxidant capacity. It was observed that l-BMAA caused a significant increase in the degree of lipid peroxidation and catalase activity in both organs. A significant increase in glutathione peroxidase activity was obtained only in liver, whereas glutathione levels were also increased in both organs. The total antioxidant capacity decreased in liver and increased in kidney. These results suggest that the oxidative stress was higher in liver than in kidney, and might be crucial for l-BMAA toxicological action.     

Role of β2-adrenoceptor blockade and circulating adrenaline level for the pressor responses to β-adrenoceptor blocking drugs in rats

Summary The present experiments were designed to elucidate what mechanism(s) would be responsible for -adrenoceptor blocking drugs (-blockers)-induced pressor responses in rats. In urethane-anaesthetized rats, 6 -blockers at i.v. doses ranging from 0.3 to 300 g/kg evoked the pressor response in a dose-dependent manner. The relative potency in causing the pressor action was correlated not to their ₁-blocking activities (r=0.374, P>0.05) but to their ₂-blocking ones (r=0.856, P<0.05). In pithed or adrenalectomized rats with low levels of plasma catecholamines, however, propranolol failed to exert its sustained pressor action. Propranolol (300 g/kg i.v.) distinctly potentiated the pressor responses not to noradrenaline but to adrenaline and to electrical stimulation of the sympathetic outflow (E.S.) in pithed rats. On the contrary, there was not any potentiation of pressor response to E.S. in pithed, adrenalectomized rats treated with propranolol (300 g/kg i.v.). In rats treated with phenoxybenzamine (5 mg/kg i.v.), adrenaline was shown to have much more potent vasodilating action resulting from ₂-stimulation than noradrenaline, the dose difference for causing the diastolic blood pressure decrease by a 25 mm Hg being almost 80 times. In pithed rats, infusion of adrenaline at the rate of 0.02 g/min caused a significant increase in plasma adrenaline level from 0.02±0.01 to 0.45±0.048 ng/ml, being close to basal level obtained in urethane-anaesthetized rats. Under this situation, propranolol (1–100 g/kg i.v.) showed a distinct pressor response in a dose-dependent fashion as observed in adrenal intact rats anaesthetized with urethane. These results strongly suggest that the pressor responses to -blockers are largely due to both their inhibitory actions on the vasodilatory ₂-adrenoceptors and to their potentiating actions on the vascular response to circulating adrenaline.     

Biased β2‐adrenoceptor signalling in heart failure: pathophysiology and drug discovery

The body is constantly faced with a dynamic requirement for blood flow. The heart is able to respond to these changing needs by adjusting cardiac output based on cues emitted by circulating catecholamine levels. Cardiac β‐adrenoceptors transduce the signal produced by catecholamine stimulation via G_s proteins to their downstream effectors to increase heart contractility. During heart failure, cardiac output is insufficient to meet the needs of the body; catecholamine levels are high and β‐adrenoceptors become hyperstimulated. The hyperstimulated β₁‐adrenoceptors induce a cardiotoxic effect, which could be counteracted by the cardioprotective effect of β₂‐adrenoceptor‐mediated G_i signalling. However, β₂‐adrenoceptor‐G_i signalling negates the stimulatory effect of the G_s signalling on cardiomyocyte contraction and further exacerbates cardiodepression. Here, further to the localization of β₁‐ and β₂‐adrenoceptors and β₂‐adrenoceptor‐mediated β‐arrestin signalling in cardiomyocytes, we discuss features of the dysregulation of β‐adrenoceptor subtype signalling in the failing heart, and conclude that G_i‐biased β₂‐adrenoceptor signalling is a pathogenic pathway in heart failure that plays a crucial role in cardiac remodelling. In contrast, β₂‐adrenoceptor‐G_s signalling increases cardiomyocyte contractility without causing cardiotoxicity. Finally, we discuss a novel therapeutic approach for heart failure using a G_s‐biased β₂‐adrenoceptor agonist and a β₁‐adrenoceptor antagonist in combination. This combination treatment normalizes the β‐adrenoceptor subtype signalling in the failing heart and produces therapeutic effects that outperform traditional heart failure therapies in animal models. The present review illustrates how the concept of biased signalling can be applied to increase our understanding of the pathophysiology of diseases and in the development of novel therapies.

Linked Articles

This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Abbreviations

ACEI

ACE inhibitors

CaMKII

Ca²⁺/calmodulin‐dependent kinase II

ct

carboxy terminus

EGFR

epidermal growth receptor

Epac

exchange protein directly activated by cAMP

G_i

inhibitory G protein

GRK

GPCR kinase

G_s

stimulatory G protein

HF

heart failure

PKA

cAMP‐dependent protein kinase

SNS

sympathetic nervous system

Tables of Links

Melatonin alleviates β-zearalenol and HT-2 toxin-induced apoptosis and oxidative stress in bovine ovarian granulosa cells

β-zearalenol (β-zol) and HT-2 are mycotoxins which cause apoptosis and oxidative stress in mammalian reproductive cells. Melatonin is an endogenous antioxidant involved in apoptosis and oxidative stress-related activities. This study investigated the effects of β-zol and HT-2 on bovine ovarian granulosa cells (BGCs), and how melatonin may counteract these effects. β-zol and HT-2 inhibited cell proliferation in a dose-dependent manner, and induced apoptosis of BGCs. They also yielded upregulation of the apoptosis-related genes Bax/Bcl-2 and Caspase3 and phosphorylation of p38MAPK. Increases in intracellular ROS were observed along with higher levels of mRNA anti-oxidation markers SOD1, SOD2, and CAT. SOD1, SOD2, malonaldehyde (MDA), and glutathione peroxidase (GSH-px) activities increased, as did the levels of SOD1 and SOD2 proteins. All of these effects were reduced or entirely attenuated in BGCs pre-treated with melatonin. Our results demonstrate that melatonin has protective effects against mycotoxin-induced apoptosis and oxidative stress in BGCs.     

Neuroprotective effects of Eriobotrya japonica against β-amyloid-induced oxidative stress and memory impairment

The generation of oxygen free radicals and oxidative damage is believed to be involved in the pathogenesis of neurodegenerative disorders. Eriobotrya japonica has been used to treat several diseases in East Asia. In this study, we investigated the protective effect of an E. japonica extract against Aβ peptide-induced oxidative stress. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay demonstrated that the E. japonica extract scavenged approximately 40% of DPPH radicals. Also, treatment of the E. japonica extract inhibited Aβ_1-42-mediated neuronal cell death. Furthermore, treatment of E. japonica extract efficiently suppressed the increase in intracellular ROS triggered by the Aβ_1-42 peptide. Importantly, mice pre-treated with the E. japonica extract showed restoration of alternation behavior and reversal of Aβ_1-42-induced memory impairment.Consequently, the E. japonica extract substantially inhibited the increase in lipid peroxidation and restored superoxide dismutase activity. These results suggest that E. japonica protects from oxidative stress and cognitive deficits induced by the Aβ peptide.     

The pharmacogenetics of β-adrenergic receptor antagonists in the treatment of hypertension and heart failure

INTRODUCTION: β-Blockers have an important therapeutic role throughout the cardiovascular continuum. However, there is considerable variation in response to these drugs, which may be related to genetic influences on their pharmacokinetics and pharmacodynamic effects. AREAS COVERED: This review focuses on genetic variations in the drug metabolizing enzymes which influence the pharmacokinetics and potentially the pharmacodynamics of some β-blockers. It also reviews the polymorphisms in the adrenergic receptors (ARs) and their related pathways which are likely to influence the responses to β-blockers. EXPERT OPINION: The CYP2D6 genotypes influence the pharmacokinetics of some β-blockers but the effects on β-blocker responses have been inconsistent and there is currently no general role for CYP2D6 genotyping prior to choosing a particular β-blocker or dose. The common polymorphisms producing changes in the β(1)-ARs, and their signaling pathways, have been associated with clinical outcomes in several studies in hypertension and heart failure. Treatment with β-blockers, especially with higher doses, appears to have greater benefits in patients with the genetic forms of the β(1)-ARs which are more responsive to both agonists and antagonists. However, current data are not sufficiently consistent to support genotyping for these polymorphisms before selecting or initiating β-blocker treatment and further study results are needed to clarify the situation.     

Pharmacological effects of concomitant administration of β-adrenoceptor blocker and agonist in normal subjects: characterization by heart rate response to exercise

The effects of a combination regimen of metoprolol and ₁-adrenoceptor agonist denopamine on resting and exercise heart rate have been studied in 10 normal volunteers. Maximal ramp upright bicycle exercise was performed three times at 1-week intervals. Two hours before each exercise test, 5 mg metoprolol plus 20 mg denopamine, 5 mg metoprolol plus a denopamine placebo, or two placebos were orally administered in a double-blind fashion.During exercise after placebo administration, heart rate increased in parallel with the exercise intensity. Compared to the placebo values, resting heart rate was significantly decreased by an average of 10 beats · min^–1 by 5 mg metoprolol, whereas it was not altered by the combination regimen. During exercise, however, both the combination regimen and metoprolol alone showed a significant negative chronotropic effect, decreasing peak exercise heart rate by an average of 14 and 21 beats · min^–1, respectively. Peak oxygen uptake was also significantly decreased by both regimens.We conclude that concomitant administration of 5 mg metoprolol and 20 mg denopamine exerts an effective -adrenoceptor blocking action during exercise but a minimal effect at rest in normal subjects. The combination regimen appears to have a favourable pharmacological profile for -adrenoceptor blocker therapy in patients with chronic heart failure.     

Curcumin and β-caryophellene attenuate cadmium quantum dots induced oxidative stress and lethality in Caenorhabditis elegans model system

Curcumin (CUR) and β-caryophellene (BCP) are well known bioactive phytomolecules which are known to reduce oxidative stress in living organisms. Therefore, the present study was envisaged to explore the possible effects of CUR and BCP in suppression of cadmium quantum dots (CdTe QDs) induced toxicity in Caenorhabditis elegans. CdTe QD are luminescent nanoparticles extensively exploited for in vivo imaging, but long term bioaccumulation confer deleterious effects on living organisms. The 24-h LC₅₀ and LC₁₀₀ of CdTe QD were found to be 18.40 μg/ml and 100 μg/ml respectively. The CdTe QD exposure elevated HSP-16.2 expression mediating induction of the stress response. The CdTe QD lethality was due to increment in ROS and decline in SOD and GST expression. The present study demonstrates improved survival in BCP (50 μM) and CUR (20 μM) treated worms by over 60% (P < 0.01) and 50% (P < 0.029) in CdTe QD (100 μg/ml) exposed worms. Furthermore, BCP and CUR attenuate oxidative stress triggered by QD. The present study for the first time demonstrates CdTe QD toxicity remediation via BCP and CUR. The future investigations can unravel underlying protective effects of phytomolceules for remediating cyotoxicolgical effects of QDs.     

Does psychological stress contribute to the development of hypertension and coronary heart disease?

Summary The role of mental stress in the development of hypertension and coronary heart disease remains uncertain. Its effects are likely to depend on an interaction of at least three factors: the nature of the stressor, its perception by the individual, and the individual's physiological susceptibility. This type of interaction is supported by animal studies. Most experimental studies in man have measured the cardiovascular response to acute laboratory stressors, on the assumption that subjects showing greater reactivity are at higher risk of developing cardiovascular disease. Experimental support for this reactivity hypothesis is limited, and the technique suffers from a number of limitations, such as an emphasis on acute rather than chronic effects, and a doubtful relevance to real-life stressors. It is possible, however, that transient surges of autonomic activity occurring in response to behavioral stimuli could trigger acute cardiac events. The role of environmental stressors can be studied by ambulatory monitoring techniques. Using the Karasek Job Content Survey (a known predictor of coronary heart disease), we have found that men in high strain jobs (defined by a combination of high demands and low control) are more likely to be hypertensive, and to show left ventricular hypertrophy than men in less stressful jobs.